Abstract
A significant portion of the genome comprises sequences such as transposable elements, which carry the potential risk of disrupting genes when they are expressed. While most organisms silence these regions to prevent their proliferation, ciliates tend to eliminate transposable elements and their remnants, internal eliminated sequences (IESs), from the somatic genome. To address this challenge, small RNA-guided nuclear crosstalk has been proposed in Paramecium tetraurelia to identify sequences for excision by comparing the fully reorganized somatic genome with the non-reorganized germline genome. However, it is unclear how precise elimination is achieved for all IESs, as only a subset of IESs relies on the small RNA pathway for efficient excision. It is evident that additional, as yet unidentified factors play a role in facilitating the recruitment of the excision machinery to IESs. Therefore, the primary objective of this study is to characterize novel proteins that contribute to IES excision. First, ISWI1, a member of the highly conserved Imitation Switch (ISWI) family of ATP-dependent chromatin remodelers, is characterized. ISWI1 is the first protein to be reported that influences the excision precision of IESs. In other eukaryotes, ISWI always relies on complex partners for its full activity. After we identified two complex partners of ISWI1, ISWI1 Complex Protein (ICOP) 1 and ICOP2, we show that ISWI1 and the ICOPs localize to the maturing somatic nuclei, where IES excision occurs. The ICOPs interact with ISWI1 both in vitro and in vivo. In knockdown experiments, all three proteins show phenotypic similarities including IES retention in the reorganized somatic genome, imprecise excision at alternative IES boundaries and alterations in the nucleosome densities on IESs, suggesting shared functionality. Additionally, we screened for novel candidates among ISWI1-associated proteins identified by mass spectrometry and characterize two paralogous PHD finger proteins: development-specific PHD finger (DevPF) 1 and DevPF2. We show that these paralogs contribute differently to IES excision. The early-expressed DevPF1 localizes into some, though not all, of the meiotic germline nuclei. In later stages, DevPF1 localizes to the maturing somatic nuclei, as does the late-expressed DevPF2. The knockdown of DevPF1 completely abolishes development-specific small RNA production, while DevPF2 knockdown mainly affects the late-produced small RNA population. We also demonstrate that DevPF1 knockdown exhibits no preference regarding IES length while in DevPF2 knockdown, preferably long IESs are retained. Taken together, I present work that characterizes five new players essential for maintaining genome integrity during nuclear maturation, adding to our picture of IES excision in Paramecium and related processes in other organisms.
